Method for evaluating filtration conditions and method for producing cell products

Abstract

A method for evaluating filtration condition(s), the method including: under conditions where cell growth is suppressed, feeding a culture fluid containing cells from a culture tank 11 containing the culture fluid to a porous membrane 12, returning the culture fluid, which has passed through the porous membrane 12 without being filtered, back to the culture tank 11 so as to circulate the culture fluid between the culture tank 11 and the porous membrane 12; and evaluating one or more filtration conditions in the porous membrane 12.

Description

[Technical Field]

[0001] This invention relates to filtration technology, specifically to a method for evaluating filtration conditions and a method for producing cell products. [Background technology]

[0002] Cell culture technology is essential for the manufacture of various biopharmaceuticals such as antibodies, growth hormones, and insulin, and has made a significant contribution to recent advances in medicine. Among biopharmaceuticals, antibody drugs, in particular, are attracting attention. Highly efficient and stable production of monoclonal antibodies by culturing antibody-producing cells is one of the industrially important themes.

[0003] Industrial cell culture methods for producing useful cell products such as antibodies are broadly classified into two types: adherent culture and suspension culture. In adherent culture, cells are attached to the inner surface of the culture vessel. In suspension culture, cells are suspended in the culture medium. Of these, suspension culture is the mainstream method due to its ease of scaling up and ease of control on a large scale.

[0004] In suspension culture methods, a system has been proposed in which, in order to culture cells in large quantities and at high density and to produce cell products continuously with high efficiency, fresh culture medium is supplied to the culture tank at a constant rate while old culture medium containing cell products is filtered and discharged from the culture tank at a constant rate. This method of culture is generally called continuous culture or perfusion culture (see, for example, Patent Documents 1 to 5). In continuous culture, the amount of culture medium supplied to the culture tank and the amount of culture medium discharged from the culture tank can be controlled to be the same. What is important in continuous culture is to efficiently separate the cells in the culture medium from the old culture medium and cell products over a long period of time, remove the old culture medium and cell products from the culture tank, and maintain the cell growth environment in the culture tank under optimal conditions for a long period of time. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Unexamined Patent Application Publication No. 2018-76291 [Patent Document 2] Japanese Unexamined Patent Application Publication No. 2009-45019 [Patent Document 3] Japanese Unexamined Patent Application Publication No. 2021-48776 [Patent Document 4] Japanese Patent No. 5696479 [Patent Document 5] Japanese Translation of PCT International Publication No. 2022-516516 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] In continuous culture, a porous membrane is used to separate cells in the culture solution in the culture tank from the old culture solution and cell products. A method for appropriately evaluating the filtration conditions in the porous membrane is desired. Therefore, one of the problems of the present invention is to provide a method for evaluating filtration conditions that can appropriately evaluate the filtration conditions of a cell culture solution and a method for producing a cell product. [Means for Solving the Problems]

[0007] [1] According to an aspect of the present invention, under conditions where cell growth is suppressed, a culture solution containing cells is sent from a culture tank containing the culture solution to a porous membrane, and the culture solution that has passed through the porous membrane without being filtered is returned to the culture tank, and the culture solution is circulated between the culture tank and the porous membrane, and one or more filtration conditions in the porous membrane are evaluated. A method for evaluating filtration conditions is provided.

[0008] [2] In the method for evaluating filtration conditions of [1] above, when returning the culture solution that has passed through the porous membrane without being filtered to the culture tank, the culture solution that has passed through the porous membrane without being filtered and the culture solution that has been filtered by the porous membrane may be returned to the culture tank.

[0009] [3] The method for evaluating the filtration conditions of [1] or [2] above may further include evaluating the filtration performance of the porous membrane under one or more filtration conditions, and based on the filtration performance of the porous membrane, one or more filtration conditions in the porous membrane may be evaluated.

[0010] [4] In the method for evaluating the filtration conditions according to any one of [1] to [3] above, the condition under which cell growth is suppressed may be a condition under which the progression of the cell cycle is suppressed.

[0011] [5] In the method for evaluating the filtration conditions according to any one of [1] to [4] above, the condition under which cell growth is suppressed may be at least one of an ambient temperature of 15°C or lower and a culture solution temperature.

[0012] [6] In the method for evaluating the filtration conditions according to any one of [1] to [5] above, at least one of an ambient temperature of 15°C or lower and a culture solution temperature may be at least one of an ambient temperature of 10°C or lower and a culture solution temperature of 5°C or lower.

[0013] [7] In the method for evaluating the filtration conditions according to any one of [1] to [6] above, the condition under which cell growth is suppressed may be a condition under which the enzyme activity of the cell is suppressed.

[0014] [8] In the method for evaluating the filtration conditions of [7] above, the enzyme may be a cyclin-dependent kinase.

[0015] [9] In the method for evaluating the filtration conditions according to any one of [1] to [8] above, the condition under which cell growth is suppressed may be the presence of an inhibitor of the cell cycle in the culture solution.

[0016]

[10] In the method for evaluating the filtration conditions according to any one of [1] to [9] above, based on the permeability of the cell product in the porous membrane, one or more filtration conditions in the porous membrane may be evaluated.

[0017]

[11] In any of the filtration condition evaluation methods described in [1] to

[10] above, one or more filtration conditions in the porous membrane may be evaluated based on the permeate flux in the porous membrane.

[0018]

[12] In any of the filtration condition evaluation methods described in [1] to

[11] above, one or more filtration conditions in the porous membrane may be evaluated based on the intermembrane pressure difference in the porous membrane.

[0019]

[13] In any of the filtration condition evaluation methods described in [1] to

[12] above, one or more filtration conditions in the porous membrane may be evaluated based on the turbidity of the filtrate.

[0020]

[14] In any of the filtration condition evaluation methods described in [1] to

[13] above, one or more filtration conditions may be one or more conditions relating to the structure, material, or physical properties of the porous membrane.

[0021]

[15] In any of the filtration condition evaluation methods described in [1] to

[14] above, one or more filtration conditions may be one or more conditions of the culture medium.

[0022]

[16] In any of the filtration condition evaluation methods described in [1] to

[15] above, one or more filtration conditions may be one or more conditions for the cell density in the culture medium.

[0023]

[17] In any of the filtration condition evaluation methods described in [1] to

[16] above, one or more filtration conditions may be one or more conditions of the flow rate of the culture medium sent to the porous membrane.

[0024]

[18] In any of the filtration condition evaluation methods described in [1] to

[17] above, one or more filtration conditions may be one or more conditions of the flow rate of the culture medium filtered through the porous membrane.

[0025]

[19] In any of the filtration condition evaluation methods described in [1] to

[18] above, one or more filtration conditions may be one or more conditions of shear stress on the liquid-contacting surface of the porous membrane due to the flow of culture medium supplied to the porous membrane.

[0026]

[20] In any of the filtration condition evaluation methods described in [1] to

[19] above, the pathway through which the culture medium, including the culture tank and the porous membrane, circulates may be located within the temperature control tank.

[0027]

[21] In any of the filtration condition evaluation methods described in [1] to

[20] above, when circulating the culture medium between the culture tank and the porous membrane, it is not necessary to perform any active operations to maintain the composition of the culture medium.

[0028]

[22] In any of the filtration condition evaluation methods described in [1] to

[21] above, when circulating the culture medium between the culture tank and the porous membrane, it is not necessary to add the culture medium from the outside to the pathway through which the culture medium circulates, including the culture tank and the porous membrane.

[0029]

[23] In any of the filtration condition evaluation methods described in [1] to

[22] above, it is not necessary to control at least one of the dissolved oxygen and pH of the culture medium when circulating the culture medium between the culture tank and the porous membrane.

[0030]

[24] In any of the filtration condition evaluation methods described in [1] to

[23] above, when circulating the culture medium between the culture tank and the porous membrane, it is not necessary to breed the cells from the pathway through which the culture medium circulates, including the culture tank and the porous membrane.

[0031]

[25] In any of the filtration condition evaluation methods described in [1] to

[24] above, when circulating the culture medium between the culture tank and the porous membrane, the culture medium may be collected for sampling from the pathway through which the culture medium circulates, including the culture tank and the porous membrane.

[0032]

[26] The method for evaluating the filtration conditions in

[25] above may further include measuring the cell density in the sampled culture medium.

[0033]

[27] The method for evaluating the filtration conditions in

[25] or

[26] above may further include measuring the viability of cells in the sampled culture medium.

[0034]

[28] Any of the filtration condition evaluation methods described in

[25] to

[27] above may further include measuring the concentration of cell products in the sampled culture medium.

[0035]

[29] Any of the filtration condition evaluation methods described in

[25] to

[28] above may further include measuring the turbidity of the sampled culture medium.

[0036]

[30] In any of the filtration condition evaluation methods described in [1] to

[29] above, the porous membrane may be a hollow fiber membrane.

[0037]

[31] In any of the filtration condition evaluation methods described in [1] to

[30] above, the porous membrane may be a microfiltration membrane.

[0038]

[32] According to an aspect of the present invention, a method for producing cell products is provided, comprising using filtration conditions to send a culture medium containing cells from a culture tank to a porous membrane under conditions in which cells proliferate, returning the culture medium that has passed through the porous membrane without being filtered back to the culture tank, recovering the culture medium containing the cell products filtered by the porous membrane, and circulating at least a portion of the culture medium between the culture tank and the porous membrane, wherein the filtration conditions are filtration conditions obtained by evaluating one or more filtration conditions in the porous membrane under conditions in which cell proliferation is suppressed, sending a culture medium containing cells from a culture tank to a porous membrane, returning the culture medium that has passed through the porous membrane without being filtered back to the culture tank, circulating the culture medium between the culture tank and the porous membrane.

[0039]

[33] In the method for producing cell products described in

[32] above, when evaluating one or more filtration conditions in a porous membrane, when returning the culture medium that has passed through the porous membrane without being filtered back to the culture tank, the culture medium that has passed through the porous membrane without being filtered and the culture medium that has been filtered back to the porous membrane may be returned to the culture tank.

[0040]

[34] In the method for producing cell products described in

[32] or

[33] above, the conditions under which cells proliferate may also be the conditions under which the cell cycle progresses.

[0041]

[35] In any of the methods for producing cell products described in

[32] to

[34] above, the conditions for cell proliferation may be at least one of an ambient temperature of 15°C or higher and a culture medium temperature.

[0042]

[36] In the method for producing cell products described in

[35] above, 15°C The above At least one of the ambient temperature and the culture medium temperature may be higher than 20°C, 25°C, 30°C, or 35°C.

[0043]

[37] In any of the methods for producing cell products described in

[32] to

[36] above, the conditions under which the cells proliferate may be conditions in which the enzymes of the cells are activated.

[0044]

[38] In the method for producing cell products described in

[37] above, the enzyme may be a cyclin-dependent kinase.

[0045]

[39] In any of the methods for producing cell products described in

[32] to

[38] above, the conditions for cell proliferation may be the absence of a cell cycle inhibitor in the culture medium.

[0046]

[40] In any of the methods for producing cell products described in

[32] to

[39] above, the conditions under which cell proliferation is suppressed may be conditions under which the progression of the cell cycle is suppressed.

[0047]

[41] In any of the methods for producing cell products described in

[32] to

[40] above, the conditions for inhibiting cell proliferation may be at least one of an ambient temperature of 15°C or lower and a culture medium temperature.

[0048]

[42] In the method for producing cell products described in

[41] above, at least one of the ambient temperature and culture medium temperature of 15°C or lower may be at least one of the ambient temperature and culture medium temperature of 10°C or lower or 5°C or lower.

[0049]

[43] In any of the methods for producing cell products described in

[32] to

[42] above, the conditions under which cell proliferation is suppressed may be conditions under which the enzyme activity of the cells is suppressed.

[0050]

[44] In the method for producing cell products described in

[43] above, the enzyme may be a cyclin-dependent kinase.

[0051]

[45] In any of the methods for producing cell products described in

[32] to

[44] above, the conditions for inhibiting cell proliferation may include the presence of a cell cycle inhibitor in the culture medium.

[0052]

[46] In any of the methods for producing cell products described in

[32] to

[45] above, one or more filtration conditions in the porous membrane may be evaluated based on the permeability of the cell products in the porous membrane.

[0053]

[47] In any of the methods for producing cell products described in

[32] to

[46] above, one or more filtration conditions in the porous membrane may be evaluated based on the permeation flux in the porous membrane.

[0054]

[48] ​​In any of the methods for producing cell products described in

[32] to

[47] above, one or more filtration conditions in the porous membrane may be evaluated based on the intermembrane pressure difference in the porous membrane.

[0055]

[49] In any of the methods for producing cell products described in

[32] to

[48] above, one or more filtration conditions in the porous membrane may be evaluated based on the turbidity of the filtrate.

[0056]

[50] In any of the methods for producing cell products described in

[32] to

[49] above, one or more filtration conditions may be one or more conditions relating to the structure, material, or physical properties of the porous membrane.

[0057]

[51] In any of the methods for producing cell products described in

[32] to

[50] above, one or more filtration conditions may be one or more conditions of the culture medium.

[0058]

[52] In any of the methods for producing cell products described in

[32] to

[51] above, one or more filtration conditions may be one or more conditions for the cell density in the culture medium.

[0059]

[53] In any of the methods for producing cell products described in

[32] to

[52] above, one or more filtration conditions may be one or more conditions of the flow rate of the culture medium sent to the porous membrane.

[0060]

[54] In any of the methods for producing cell products described in

[32] to

[53] above, one or more filtration conditions may be one or more conditions of the flow rate of the culture medium filtered through the porous membrane.

[0061]

[55] In any of the methods for producing cell products described in

[32] to

[54] above, one or more filtration conditions may be one or more conditions of shear stress on the liquid-contacting surface of the porous membrane due to the flow of culture medium supplied to the porous membrane.

[0062]

[56] In any of the methods for producing cell products described in

[32] to

[55] above, when evaluating one or more filtration conditions in a porous membrane, a pathway through which the culture medium circulates, including a culture tank and a porous membrane, may be placed inside a temperature-controlled tank.

[0063]

[57] In any of the methods for producing cell products described in

[32] to

[56] above, when evaluating multiple filtration conditions in a porous membrane, it is not necessary to perform any active operations to maintain the composition of the culture medium by circulating the culture medium between the culture tank and the porous membrane.

[0064]

[58] In any of the methods for producing cell products described in

[32] to

[57] above, when evaluating multiple filtration conditions in a porous membrane, when circulating the culture medium between the culture tank and the porous membrane, it is not necessary to add culture medium from the outside to the pathway through which the culture medium circulates, including the culture tank and the porous membrane.

[0065]

[59] In any of the methods for producing cell products described in

[32] to

[58] above, when evaluating multiple filtration conditions in a porous membrane, it is not necessary to control at least one of the dissolved oxygen and pH of the culture medium when circulating the culture medium between the culture tank and the porous membrane.

[0066]

[60] In any of the above methods for producing cell products described in

[32] to

[59] , when evaluating multiple filtration conditions in a porous membrane, it is not necessary to bleed the cells from the pathway through which the culture medium circulates, including the culture tank and the porous membrane, when circulating the culture medium between the culture tank and the porous membrane.

[0067]

[61] In any of the methods for producing cell products described in

[32] to

[60] above, when evaluating one or more filtration conditions in a porous membrane, the culture medium may be circulated between the culture tank and the porous membrane, and the culture medium may be collected for sampling from the pathway through which the culture medium circulates, including the culture tank and the porous membrane.

[0068]

[62] The method for producing cell products described in

[61] above may further include measuring the density of cells in the sampled culture medium.

[0069]

[63] The method for producing cell products described in

[61] or

[62] above may further include measuring the viability of cells in a sampled culture medium.

[0070]

[64] Any of the cell production methods described in

[61] to

[63] above may further include measuring the concentration of the cell product in the sampled culture medium.

[0071]

[65] Any of the cell production methods described in

[61] to

[64] above may further include measuring the turbidity of the sampled culture medium.

[0072]

[66] In any of the methods for producing cell products described in

[32] to

[65] above, the porous membrane may be a hollow fiber membrane.

[0073]

[67] In any of the methods for producing cell products described in

[32] to

[66] above, the porous membrane may be a microfiltration membrane.

[0074]

[68] In any of the methods for producing cell products described in

[32] to

[67] above, the cells may be perfused. [Effects of the Invention]

[0075] According to the present invention, it is possible to provide a method for evaluating filtration conditions that can appropriately assess the filtration conditions of a cell culture medium, and a method for producing cell products. [Brief explanation of the drawing]

[0076] [Figure 1] This is a schematic diagram showing an evaluation system for the filtration conditions of a hollow fiber membrane according to an embodiment. [Figure 2] This is a schematic diagram showing a production system for cell products according to an embodiment. [Figure 3] This graph shows the time change in cell density in the culture medium circulating between the culture tank and the hollow fiber membrane in the example. [Figure 4]This graph shows the time change in cell density in the culture medium circulating between the culture tank and the hollow fiber membrane in the example. [Figure 5] This graph shows the time-dependent change in cell viability in the culture medium circulating between the culture tank and the hollow fiber membrane in the example. [Figure 6] This graph shows the time-dependent change in cell viability in the culture medium circulating between the culture tank and the hollow fiber membrane in the example. [Figure 7] This graph shows the time change in antibody concentration in the culture medium circulating between the culture tank and the hollow fiber membrane in the example. [Figure 8] This graph shows the time change in antibody concentration in the culture medium circulating between the culture tank and the hollow fiber membrane in the example. [Figure 9] This graph shows the time change in cell density in the culture medium circulating between the culture tank and the hollow fiber membrane in the comparative example. [Figure 10] This graph shows the time-dependent change in cell viability in the culture medium circulating between the culture tank and the hollow fiber membrane in the comparative example. [Figure 11] This graph shows the time change in antibody concentration in the culture medium circulating between the culture tank and the hollow fiber membrane in the comparative example. [Figure 12] This graph shows the time-dependent change in cell density in the culture medium within a culture vessel, as shown in the reference example. [Figure 13] This graph shows the time-dependent change in cell viability in the culture medium within a culture vessel, as shown in the reference example. [Figure 14] This graph shows the time-dependent change in antibody concentration in the culture medium within a culture vessel, as per the reference example. [Figure 15] This graph shows the time-dependent change in cell density in the culture medium within a culture vessel, as shown in the reference example. [Figure 16] This graph shows the time-dependent change in cell viability in the culture medium within a culture vessel, as shown in the reference example. [Figure 17] This graph shows the time-dependent change in antibody concentration in the culture medium within a culture vessel, as per the reference example. [Figure 18]This graph shows the time-dependent change in cell density in the culture medium within a culture vessel, as shown in the reference example. [Figure 19] This graph shows the time-dependent change in cell viability in the culture medium within a culture vessel, as shown in the reference example. [Figure 20] This graph shows the time-dependent change in antibody concentration in the culture medium within a culture vessel, as per the reference example. [Modes for carrying out the invention]

[0077] The following describes in detail an embodiment for carrying out the present invention (hereinafter referred to as "this embodiment"). This embodiment is provided to facilitate understanding of the present invention and is not intended to limit its interpretation. The present invention is not limited to this embodiment and can be implemented in various modifications within the scope of its gist.

[0078] Referring to Figure 1, the method for evaluating filtration conditions according to this embodiment includes: sending the culture medium from a culture tank 11 containing the culture medium containing cells to a porous membrane 12 under conditions that suppress cell proliferation; returning the culture medium that has passed through the porous membrane 12 without being filtered back to the culture tank 11; circulating the culture medium between the culture tank 11 and the porous membrane 12; evaluating the filtration performance of the porous membrane 12 under one or more filtration conditions; and evaluating one or more filtration conditions in the porous membrane 12 based on the filtration performance of the porous membrane 12. When returning the culture medium that has passed through the porous membrane 12 without being filtered back to the culture tank 11, it is also possible to return both the culture medium that has passed through the porous membrane 12 without being filtered and the culture medium that has been filtered by the porous membrane 12 to the culture tank 11. However, this does not prevent the recovery of the culture medium without returning the entire amount of culture medium to the culture tank 11, as long as the same amount of culture medium as the recovered culture medium is added to the culture medium in the culture tank 11. The recovery of the culture medium may also be performed by recovering the culture medium filtered by the porous membrane 12 without returning all or part of it to the culture tank 11. The amount of culture medium to be recovered is not limited, and fluctuations in cell density and product concentration in the culture medium within the culture vessel 11 are suppressed by replenishing the system with the same amount of culture medium. This recovery is distinct from the sampling described later, which is for the purpose of analyzing the culture medium.

[0079] The culture tank 11 is, for example, a container having an internal space sealed off from the outside air. However, the culture tank 11 may be provided with vents or filters to maintain a constant internal pressure. The vents or filters preferably have a pore size of 0.2 μm or less to prevent external bacteria from entering the culture tank 11. The culture tank 11 may be provided with a stirring device 16 for stirring the culture medium inside the culture tank 11. Cells are cultured in suspension in the culture medium inside the culture tank 11.

[0080] The cells cultured in the culture vessel 11 are not particularly limited. The cells may be of animal origin, including human, or of microorganism origin. The cells may be eukaryotic or prokaryotic. Examples of animals include mammals, reptiles, birds, amphibians, fish, and insects. The cells may be genetically modified cells. Examples of cells include CHO (Chinese Hamster Ovary) cells, HEK cells, BHK-21 cells, Sp2 / 0 cells, SP2 / 0-Ag14 cells, NS0 cells, Vero cells, PER.C6 cells, yeast, Bacillus subtilis, and Escherichia coli.

[0081] Cells produce and release into the culture medium products that can be used as pharmaceuticals, for example. Examples of pharmaceutical products include peptides, proteins, and viruses (including virus-like particles). Examples of proteins include antibodies, hormones, cytokines, growth factors, enzymes, and plasma proteins. Proteins may also be recombinant proteins.

[0082] The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may be a human antibody, or an antibody protein derived from mammals other than humans, such as cattle and mice. Alternatively, the antibody may be a chimeric antibody protein with human IgG, or a humanized antibody. A chimeric antibody with human IgG is an antibody in which the variable region is derived from a non-human organism such as a mouse, but the other constant regions are replaced with human-derived immunoglobulins. A humanized antibody is an antibody in which the complementarity-determining region (CDR) of the variable region is derived from a non-human organism, but the other framework regions (FR) are derived from humans. Humanization further reduces immunogenicity compared to chimeric antibodies.

[0083] The shape of the porous membrane 12 is not particularly limited. Examples of porous membranes include hollow fiber membranes, flat membranes, and tubular membranes. In the following, an example in which the porous membrane 12 is a hollow fiber membrane will be described. In the porous membrane 12, the surface to which the culture medium is supplied is called the primary side of the hollow fiber membrane. The surface from which the permeate that has permeated through the hollow fiber membrane flows out is called the secondary side of the hollow fiber membrane. In an embodiment in which the culture medium to be filtered is supplied to the inner circumferential surface, the inner circumferential surface of the hollow fiber membrane is the primary side, and the outer circumferential surface of the hollow fiber membrane is the secondary side. In an embodiment in which the culture medium to be filtered is supplied to the outer circumferential surface, the outer circumferential surface of the hollow fiber membrane is the primary side, and the inner circumferential surface of the hollow fiber membrane is the secondary side.

[0084] The types of porous membranes 12 are not particularly limited. Examples of porous membranes include coarse filtration membranes, microfiltration membranes, ultrafiltration membranes, dialysis membranes, nanofiltration membranes, reverse osmosis membranes, and forward osmosis membranes.

[0085] The filtration method in the porous membrane 12 may be a tangential flow filtration (TFF) method. A tangential flow filtration method is a filtration method in which the culture medium is flowed in a direction parallel to the primary surface of the hollow fiber membrane. The tangential flow filtration method includes the alternating tangential flow filtration (ATF) method. In this embodiment, when simply referred to as a tangential flow filtration (TFF) method, it may refer to a filtration method in which the culture medium is flowed in one direction on the primary surface of the hollow fiber membrane. An alternating tangential flow filtration (ATF) method refers to a filtration method in which the culture medium is flowed back and forth on the primary surface of the hollow fiber membrane.

[0086] In the example shown in Figure 1, a channel 13 for sending the culture medium from the culture tank 11 to the porous membrane 12, a channel 14 for returning the culture medium that has passed through the porous membrane 12 without being filtered back to the culture tank 11, and optionally a channel 15 for returning the culture medium filtered by the porous membrane 12 back to the culture tank 11. The culture medium flowing through channel 13 may contain cells and cell products. The culture medium flowing through channel 14 that has passed through the porous membrane 12 without being filtered may contain cells and cell products. The culture medium flowing through channel 15 that has been filtered by the porous membrane 12 may contain cell products.

[0087] The channel 13 is equipped with a pump 23 for, for example, sending the culture medium in the culture tank 11 to the porous membrane 12. Examples of pumps include, but are not limited to, diaphragm pumps, tubular pumps, centrifugal pumps, and rotary pumps. The channel 13 may be equipped with a pressure gauge 33 for measuring the pressure of the culture medium supplied to the porous membrane 12. The channel 13 may be equipped with a flow meter for measuring at least one of the flow velocity and flow rate of the culture medium flowing through the channel 13. The channel 13 may be equipped with a thermometer for measuring the temperature of the culture medium flowing through the channel 13. The channel 13 may be equipped with a sampling section for sampling the culture medium flowing through the channel 13. The sampling section is closed when not sampling.

[0088] The channel 14 may be equipped with a pump for sending the culture medium that has not been filtered by the porous membrane 12, passing through the hollow portion without passing through the pore portion of the porous membrane 12, to the culture tank 11. The pump may be provided in both the channel 13 and the channel 14, or in either one. The channel 14 may be equipped with a pressure gauge 34 for measuring the pressure of the culture medium that has passed through the porous membrane 12. The channel 14 may be equipped with a flow meter for measuring at least one of the flow velocity and flow rate of the culture medium flowing through the channel 14. The channel 14 may be equipped with a thermometer for measuring the temperature of the culture medium flowing through the channel 14. The channel 14 may be equipped with a sampling section for sampling the culture medium flowing through the channel 14. The sampling section is closed when not sampling.

[0089] The flow path 15 is equipped with a pump 25 for sending the culture medium filtered by the porous membrane 12, for example, through the pores of the porous membrane 12, to the culture tank 11. The flow path 15 may also be equipped with a pressure gauge 35 for measuring the pressure of the culture medium filtered by the porous membrane 12. The flow path 15 may also be equipped with a flow meter for measuring at least one of the flow velocity and flow rate of the culture medium flowing through the flow path 15. The flow path 15 may also be equipped with a sampling section for sampling the culture medium flowing through the flow path 15. The sampling section is closed when not sampling.

[0090] The culture tank 11 and the porous membrane 12 form at least part of the pathway through which the culture medium circulates. Furthermore, the channels 13, 14, and 15 also form at least part of the pathway through which the culture medium circulates. The pathway through which the culture medium circulates may be closed off from the outside, but as described above, the culture tank 11 may be provided with vents or filters to maintain a constant internal pressure.

[0091] The culture vessel 11 and the porous membrane 12 may be placed inside a temperature control chamber 50 that controls the internal temperature. The channels 13, 14, and 15 may also be placed inside the temperature control chamber 50. The temperature control chamber 50 controls the ambient temperature of the culture vessel 11, the porous membrane 12, and the channels 13, 14, and 15. The temperature control chamber 50 may be a constant temperature bath or refrigerator, or it may be a booth, room, or building that maintains a constant temperature.

[0092] The conditions under which cell proliferation is suppressed are not particularly limited, but for example, conditions under which the progression of the cell cycle is suppressed. Conditions under which the progression of the cell cycle is suppressed include, for example, a low ambient temperature of 15°C or less in the culture vessel 11 and porous membrane 12, or a temperature of 15°C or less in the culture medium. The ambient temperature or the temperature of the culture medium is preferably 10°C or less, or 5°C or less. From the viewpoint of suppressing cell death, it is preferable that the ambient temperature of the culture vessel 11 and porous membrane 12, or the temperature of the culture medium, be 0°C or higher, 1°C or higher, 2°C or higher, or 3°C or higher. The temperature of the culture medium may be measured directly, or it may be estimated and measured from the ambient temperature of the culture vessel 11 and porous membrane 12. Based on the measured temperature of the culture medium, the temperature of the culture medium may be directly controlled by a temperature control device, or the ambient temperature of the culture vessel 11 and porous membrane 12 may be controlled by a temperature control device. Conditions under which the progression of the cell cycle is suppressed may also include, for example, conditions under which the activity of enzymes such as cyclin-dependent kinases in cells is suppressed. Conditions that suppress cell cycle progression may include the presence of cell cycle inhibitors in the culture medium. Under conditions that suppress cell proliferation, cell activity decreases, cell cycle progression is suppressed, cell division is inhibited, and the production of products is also suppressed.

[0093] In this embodiment, both the culture medium that has passed through the porous membrane 12 without filtration and the culture medium that has been filtered by the porous membrane 12 may be returned to the culture tank 11. In this case, under conditions in which cell proliferation is suppressed, the total amount of cells and the total amount of products in the system are maintained, and fluctuations in the cell density and product concentration in the culture medium circulating between the culture tank 11 and the porous membrane 12 are suppressed.

[0094] Under conditions where cell proliferation is suppressed, cell activity decreases, and the amount of culture medium components required by the cells also decreases, so it is not necessary to add culture medium from the outside to the circulation pathway of the culture medium. Furthermore, by not adding culture medium, fluctuations in the cell density and the concentration of cell products in the culture medium are suppressed. Moreover, under conditions where cell proliferation is suppressed, cell activity decreases, and fluctuations in dissolved oxygen concentration (DO) and pH in the culture medium are suppressed, so it is not necessary to control the dissolved oxygen concentration and pH by adding gases such as oxygen or carbon dioxide from the outside to the circulation pathway of the culture medium. Furthermore, under conditions where cell proliferation is suppressed, the cell density in the culture medium becomes almost constant, so it is not necessary to breed cells out of the circulation pathway of the culture medium.Therefore, according to the evaluation method of filtration conditions according to this embodiment, it is possible to simplify the operations for cell culture.However, for example, a portion of the culture medium may be sampled to monitor cell density, cell viability, and the concentration of cell products.Sampling is not intended to adjust the cell density and is therefore distinct from breeding.

[0095] One or more filtration conditions in the porous membrane 12 are evaluated based on the filtration performance of the porous membrane 12. The filtration performance of the porous membrane 12 may be expressed, for example, by at least one of the following: high permeability of cell products in the porous membrane 12, high shielding rate of impurities in the porous membrane 12, low intermembrane pressure difference in the porous membrane 12, large volume of liquid that can be filtered before the intermembrane pressure difference in the porous membrane 12 rises, large volume of liquid that can be filtered before the permeation flux in the porous membrane 12 begins to decrease, low pressure loss in the porous membrane 12, large flow rate in the porous membrane 12, high flow velocity in the porous membrane 12, and high permeation flux rate in the porous membrane 12.

[0096] One or more filtration conditions in the porous membrane 12 are evaluated based on whether the filtration performance of the porous membrane 12 meets a predetermined standard or is relatively superior. Alternatively, at least some of the filtration conditions in the porous membrane 12 may be selected based on whether the filtration performance of the porous membrane 12 meets a predetermined standard or is relatively superior.

[0097] If the cell density and the concentration of products in the culture medium supplied to the porous membrane 12 fluctuate, it may become difficult to accurately evaluate the filtration performance of the porous membrane 12. In contrast, as described above, in this embodiment, cell proliferation is suppressed, and fluctuations in the cell density and the concentration of products in the culture medium are suppressed. When the culture medium filtered by the porous membrane 12 is returned to the culture tank 11, fluctuations in the cell density and the concentration of products in the culture medium are further suppressed. Therefore, according to this embodiment, it is possible to evaluate the filtration performance of the porous membrane 12 while keeping the cell density and the concentration of products in the culture medium substantially constant. Furthermore, when the culture medium filtered by the porous membrane 12 is returned to the culture tank 11, it may be possible to omit the replenishment of culture medium to the culture tank 11.

[0098] One or more filtration conditions are, for example, one or more conditions of the structure of the porous membrane 12. Conditions of the structure of the porous membrane 12 include, for example, film thickness, inner diameter, outer diameter, membrane surface opening ratio, porosity, pore diameter, average pore diameter, pore diameter variation, blocked pore diameter, pore diameter to fiber diameter ratio, membrane surface anisotropy, membrane pore anisotropy, membrane surface roughness, and change in pore diameter from the primary side to the secondary side. For example, multiple porous membranes 12 having various structures are prepared, and the filtration performance of each porous membrane 12 is evaluated. Furthermore, it is evaluated whether one or more conditions of the structure of the porous membrane 12 provide filtration performance that meets a predetermined standard, or whether they are relatively superior.

[0099] One or more filtration conditions are, for example, one or more conditions for the material of the porous membrane 12. For example, multiple porous membranes 12 made of various materials are prepared, and the filtration performance of each porous membrane 12 is evaluated. Furthermore, it is evaluated whether one or more conditions for the material of the porous membrane 12 provide filtration performance that meets a predetermined standard, or whether they are relatively superior.

[0100] One or more filtration conditions are, for example, one or more conditions of the physical properties of the porous membrane 12. Conditions of the physical properties of the porous membrane 12 include, for example, hydrophilicity, hydrophobicity, cationicity, anionicity, elastic limit pressure, and bubble point. For example, multiple porous membranes 12 having various physical properties are prepared, and the filtration performance of each porous membrane 12 is evaluated. Furthermore, it is evaluated whether one or more conditions of the physical properties of the porous membrane 12 provide filtration performance that meets a predetermined standard, or whether they are relatively superior.

[0101] One or more filtration conditions are, for example, one or more conditions of the culture medium. Conditions of the culture medium include, for example, the contents of the culture medium, the concentration of each component in the culture medium, the viscosity of the culture medium, the pH of the culture medium, the electrical conductivity of the culture medium, and the turbidity of the culture medium. Examples of contents of the culture medium include the composition of the culture medium, antifoaming agents, salts, nucleic acids such as DNA and RNA, host cell-derived proteins (HCPs), lipids, and polysaccharides. For example, multiple culture mediums with different conditions are prepared, and the filtration performance of the porous membrane 12 is evaluated when using each culture medium. Furthermore, it is evaluated whether one or more conditions of the culture medium provide filtration performance that meets a predetermined standard, or whether they are relatively superior.

[0102] One or more filtration conditions are, for example, multiple conditions for the cell density in the culture medium. For example, multiple culture media with different cell densities are prepared, and the filtration performance of the porous membrane 12 is evaluated when using each culture medium. Furthermore, it is evaluated whether one or more conditions for cell density provide filtration performance that meets a predetermined standard, or whether it is relatively superior.

[0103] One or more filtration conditions are, for example, one or more conditions of the flow rate of the culture medium supplied to the porous membrane 12. For example, the filtration performance of the porous membrane 12 is evaluated when the culture medium is supplied to the porous membrane 12 at different flow rates. Furthermore, it is evaluated whether one or more conditions of the flow rate of the culture medium supplied to the porous membrane 12 provide filtration performance that meets a predetermined standard, or whether it is relatively superior. The flow rate may be expressed as volumetric flow rate, mass flow rate, linear velocity, or shear rate at the surface of the porous membrane 12.

[0104] One or more filtration conditions are, for example, one or more conditions for the flow rate of the culture medium filtered through the porous membrane 12. For example, the culture medium filtered through the porous membrane 12 is delivered at different flow rates, and the filtration performance of the porous membrane 12 is evaluated for each flow rate. Furthermore, it is evaluated whether one or more conditions for the flow rate of the culture medium filtered through the porous membrane 12 provide filtration performance that meets a predetermined standard, or whether it is relatively superior.

[0105] One or more filtration conditions are, for example, one or more conditions of shear stress at the liquid-contacting surface of the porous membrane 12. The liquid-contacting surface is the surface of the porous membrane 12 that comes into contact with the liquid being filtered. If the porous membrane 12 is a hollow fiber membrane, it is the primary side surface. For example, the filtration performance of the porous membrane 12 is evaluated by changing the shear stress at the liquid-contacting surface of the porous membrane 12 and using each shear stress. Furthermore, it is evaluated whether one or more conditions of shear stress at the liquid-contacting surface of the porous membrane 12 provide filtration performance that meets a predetermined standard, or whether it is relatively superior.

[0106] According to the filtration condition evaluation method of this embodiment, it is possible to select one or more filtration conditions that allow the porous membrane 12 to exhibit filtration performance that meets predetermined standards while suppressing fluctuations in the cell density and product concentration contained in the culture medium circulating between the culture tank 11 and the porous membrane 12.

[0107] Next, referring to Figure 2, the method for producing cell products according to this embodiment includes, under conditions in which cells proliferate, using the filtration conditions obtained by the filtration condition evaluation method described above, sending the culture medium from a culture tank 111 containing the culture medium containing cells to a porous membrane 112, returning the culture medium that has passed through the porous membrane 112 without being filtered back to the culture tank 111, recovering the culture medium containing the cell products filtered by the porous membrane 112, and circulating at least a portion of the culture medium between the culture tank 111 and the porous membrane 112.

[0108] The conditions under which cells proliferate are not particularly limited as long as the cells produce products and the products are continuously supplied to the culture medium, but for example, they are conditions under which the cell cycle progresses. Conditions under which the cell cycle progresses include, for example, an ambient temperature of 15°C or higher in the culture vessel 111 and porous membrane 112, or a temperature of 15°C or higher in the culture medium. The ambient temperature or the temperature of the culture medium is preferably higher than 20°C, 25°C, 30°C, or 35°C. Furthermore, from the viewpoint of suppressing cell death, the ambient temperature of the culture vessel 111 and porous membrane 112 or the temperature of the culture medium is preferably 60°C or lower, 50°C or lower, or 40°C or lower. The temperature of the culture medium may be measured directly, or it may be estimated and measured from the ambient temperature of the culture vessel 111 and porous membrane 112. Based on the measured temperature of the culture medium, the temperature of the culture medium may be directly controlled by a temperature control device, or the ambient temperature of the culture vessel 111 and porous membrane 112 may be controlled by a temperature control device. The conditions under which the cell cycle progresses may include, for example, the activation of enzymes such as cyclin-dependent kinases in the cells. Alternatively, the conditions under which the cell cycle progresses may include the absence of cell cycle inhibitors in the culture medium.

[0109] The cells cultured in the culture vessel 111 are preferably the same as the cells used in the filtration condition evaluation method, but they may be different. Furthermore, the products of the cells filtered through the porous membrane 112 are preferably the same as the products of the cells filtered in the filtration condition evaluation method, but they may be different.

[0110] Between the culture tank 111 and the porous membrane 112, there is a channel 113 for sending the culture medium from the culture tank 111 to the porous membrane 112, and a channel 114 for returning the culture medium that has passed through the porous membrane 112 without being filtered by the porous membrane 112 back to the culture tank 111. The culture medium flowing through channel 113 may contain cells and cell products. The culture medium flowing through channel 114, which has passed through the hollow portion of the porous membrane 112 without passing through the pore portion and has not been filtered by the porous membrane 112, may contain cells and cell products. In addition, a channel 115 is connected to the porous membrane 112 for recovering the culture medium filtered by the porous membrane 112. The culture medium flowing through channel 115, which has passed through the pore portion of the porous membrane 112 and been filtered by the porous membrane 112, may contain cell products. The culture medium filtered by the porous membrane 112 is recovered, for example, in a container 201. The container 201 may be aseptically connected to the channel 115. The channel 115, through which the culture medium filtered by the porous membrane 112 flows, may be directly connected, for example, to a column used for the next purification step. The culture medium filtered by the porous membrane 112 is not returned to the culture vessel 111.

[0111] The channel 113 is equipped with, for example, a pump 123 for sending the culture medium from the culture tank 111 to the porous membrane 112. The channel 113 may also be equipped with a pressure gauge 133 for measuring the pressure of the culture medium supplied to the porous membrane 112. The channel 113 may also be equipped with a flow meter for measuring at least one of the flow velocity and flow rate of the culture medium flowing through the channel 113. The channel 113 may also be equipped with a thermometer for measuring the temperature of the culture medium flowing through the channel 113. The channel 113 may also be equipped with a sampling section for sampling the culture medium flowing through the channel 113. The sampling section is closed when not sampling.

[0112] The channel 114 may be equipped with a pump for sending the culture medium that has not been filtered by the porous membrane 112, passing through the hollow portion without passing through the pore portion of the porous membrane 112, to the culture tank 111. The pump may be provided in both the channel 113 and the channel 114, or in either one. The channel 114 may be equipped with a pressure gauge 134 for measuring the pressure of the culture medium that has passed through the porous membrane 112. The channel 114 may be equipped with a flow meter for measuring at least one of the flow velocity and flow rate of the culture medium flowing through the channel 114. The channel 114 may be equipped with a thermometer for measuring the temperature of the culture medium flowing through the channel 114. The channel 114 may be equipped with a sampling section for sampling the culture medium flowing through the channel 114. The sampling section is closed when not sampling.

[0113] The channel 115 is equipped with, for example, a pump 125 for transporting the culture medium filtered through the porous membrane 112. The channel 115 may also be equipped with a pressure gauge 135 for measuring the pressure of the culture medium filtered through the porous membrane 112. The channel 115 may also be equipped with a flow meter for measuring at least one of the flow velocity and flow rate of the culture medium flowing through the channel 115. The channel 115 may also be equipped with a sampling section for sampling the culture medium flowing through the channel 115. The sampling section is closed when not sampling.

[0114] The culture vessel 111 and the porous membrane 112 form at least part of the pathway through which the culture medium circulates. Furthermore, the channels 113 and 114 also form at least part of the pathway through which the culture medium circulates.

[0115] A flow path 116 for supplying culture medium to the culture tank 111 may be connected to the culture tank 111. The flow path 116 is connected, for example, to a culture medium tank 216 that contains the culture medium. The flow path 116 is equipped with, for example, a pump 126 for sending the culture medium to the culture tank 111. For example, pumps 125 and 126 are controlled so that the amount of culture medium that is filtered by the porous membrane 112 and does not return to the culture tank 111 is the same as the amount of culture medium supplied to the culture tank 111. This control may be performed by observing the liquid level using a liquid level sensor (level sensor) installed in the culture tank 111 and ensuring that the liquid level remains constant, or by measuring the weight of the entire culture tank 111 containing the culture medium and ensuring that the weight remains constant.

[0116] The culture tank 111 may be connected to a channel 117 for supplying air containing carbon dioxide to the culture tank 111. The channel 117 may be connected to, for example, a container 217 that contains air containing carbon dioxide. The culture tank 111 may also be connected to a channel 118 for supplying oxygen to the culture tank 111. The channel 118 may be connected to, for example, a container 218 that contains oxygen.

[0117] The culture vessel 111 may be connected to a channel 119 for draining at least a portion of the cells within the culture vessel 111. For example, by draining at least a portion of the cells within the culture vessel 111 using the channel 119, the cell density in the culture medium within the culture vessel 111 is kept constant. This prevents an increase in cell density, which can lead to a deficiency of oxygen and culture medium components in the culture medium, or an increase in the concentration of impurities. Draining at least a portion of the cells within the culture vessel 111 is called bleeding.

[0118] The culture vessel 111 may be connected to a thermometer for measuring the temperature of the culture medium inside the culture vessel 111, a DO meter for measuring dissolved oxygen (DO), or a pH meter for measuring pH.

[0119] According to the method for producing cell products of this embodiment, since the cell products are filtered using pre-established filtration conditions, it is possible to efficiently obtain the cell products.

[0120] (Example 1) After thawing the monoclonal antibody-producing Chinese hamster ovary (CHO) cell line (ATCC CRL-12445), which had been acclimatized to serum-free medium and suspended in suspension, the cells were selected and frozen. Then, 10 mL of serum-free medium with the composition shown in Table 1 was pre-dispensed into a 125 mL Erlenmeyer flask, and the cells and medium were mixed in the flask. Cell count was confirmed using a viable and dead cell autoanalyzer (Vi-CELL XR, Beckman), and the cell density was 3.5 × 10⁶. 5 The cells were diluted with culture medium to a concentration of cells / mL. Then, the cells were cultured in an incubator at 37°C under a 5% CO2 atmosphere with shaking for 4 days. [Table 1]

[0121] Four days after thawing the cells, 3.5 × 10 5 50 mL of culture medium containing cells at a density of cells / mL was placed in two new 125 mL Erlenmeyer flasks, and the cells were cultured with shaking in an incubator at 37°C under a 5% CO2 atmosphere for 3 days. After 3 days, 3.5 × 10⁶ cells were observed. 5 120 to 130 mL of culture medium containing cells at a density of cells / mL was placed into two new 250 mL Erlenmeyer flasks, and the cells were cultured with shaking in an incubator at 37°C under a 5% CO2 atmosphere for 3 days. After 3 days, 3.5 × 10⁶ cells were observed. 5 500 mL of culture medium containing cells at a density of cells / mL was placed into three new 1 L Erlenmeyer flasks, and the cells were cultured in an incubator at 37°C under a 5% CO2 atmosphere with shaking for 3 days.

[0122] In a 12L capacity culture tank that has been pre-sterilized by autoclaving, place 5.5 x 10 5Six liters of culture medium containing cells at a density of cells / mL were aseptically added. The cells were cultured for 3 days at 37°C under a 5% CO2 atmosphere, while oxygen was blown in to ensure that the DO did not fall below 70%. After 3 days, serum-free medium with the composition shown in Table 2 was added to the culture vessel at a rate of 0.28 mL / min for 3 days. [Table 2]

[0123] Next, 1.5 × 10 7 The culture medium containing cells at a density of cells / mL was aseptically collected, and 600 mL of the culture medium was transferred to a spinner flask, which had been pre-sterilized by autoclaving, to serve as a culture vessel. The membrane area of ​​the wetted part was 3 cm². 2 A mini-module of a porous hollow fiber membrane (Asahi Kasei Medical, BioOptimal MF-SL, pore size 0.4 μm) was prepared with an adjusted effective length to achieve the desired result. The spinner flask and the first opening of the hollow section on the primary side of the porous hollow fiber membrane were connected by a first channel, the second opening of the hollow section of the porous hollow fiber membrane and the spinner flask were connected by a second channel, and the secondary side of the porous hollow fiber membrane and the spinner flask were connected by a third channel.

[0124] The ambient temperature of the spinner flask and porous hollow fiber membrane was set to 4°C, and the culture medium was stirred in the spinner flask. The culture medium was delivered from the spinner flask to the porous hollow fiber membrane via a first channel using a magnetic levitation centrifugal pump (Levitronix PuraLev i30SU) and tangential flow filtration was performed. The culture medium that passed through the hollow portion of the porous hollow fiber membrane without being filtered was returned to the spinner flask via a second channel. The culture medium filtered through the porous hollow fiber membrane was returned to the spinner flask via a third channel. The first and third channels were designed to allow sampling of the culture medium inside.

[0125] Single-use pressure gauges (manufactured by PendoTECH, PREPS-N-000 or PREPS-N-012) were installed on the primary and secondary sides of the porous hollow fiber membrane, and the transmembrane pressure difference (TMP) was measured over time. The viscosity of the culture solution was measured with an EMS viscometer (EMS-1000S), and the flow rate of the culture solution from the spinner flask to the porous hollow fiber membrane was set so that the shear stress on the inner surface of the porous hollow fiber membrane was 4.58 N / m 2 The filtration flow rate in the porous hollow fiber membrane was set to 5 μL / min to be constant (1 LMH) by a pump.

[0126] Once or twice a day, the culture solution circulating between the spinner flask and the porous hollow fiber membrane was sampled, and the cell density, cell viability, and antibody concentration in the culture solution were measured. As a result, as shown in FIGS. 3, 5, and 7, the cell density, cell viability, and antibody concentration in the culture solution were each substantially constant. When the culture solution was filtered at 300 L / m with BioOptimal MF-SL, the monoclonal antibody permeation rate in BioOptimal MF-SL was 78.8%, and the transmembrane pressure difference was 43.4 kPa. 2 The monoclonal antibody permeation rate in BioOptimal MF-SL was 78.8%, and the transmembrane pressure difference was 43.4 kPa at the time when the culture solution was filtered at 300 L / m with BioOptimal MF-SL.

[0127] (Example 2) A porous hollow fiber membrane mini-module with an effective length adjusted so that the membrane area of the wetted part was 3 cm 2 was used. The same method as in Example 1 was carried out except that a porous hollow fiber membrane manufactured by Asahi Kasei, Microza UMP, with a retention pore size of 0.2 μm was used. As a result, as shown in FIGS. 3, 5, and 7, the cell density, cell viability, and antibody concentration in the culture solution were each substantially constant. When the culture solution was filtered at 300 L / m with MICROZA UMP, the monoclonal antibody permeation rate in MICROZA UMP was 80.2%, and the transmembrane pressure difference was 81.0 kPa. 2 The monoclonal antibody permeation rate in MICROZA UMP was 80.2%, and the transmembrane pressure difference was 81.0 kPa at the time when the culture solution was filtered at 300 L / m with MICROZA UMP.

[0128] (Example 3) The membrane area of the wetted part was 3 cm 2The same procedure as in Example 1 was followed, except that a porous hollow fiber membrane with a pore size of 0.65 μm, manufactured by Asahi Kasei, was used as a minimodule of porous hollow fiber membrane with an effective length adjusted to achieve the desired result. As a result, as shown in Figures 3, 5, and 7, the cell density, cell viability, and antibody concentration in the culture medium were approximately constant. The culture medium was 300 L / m³ with MICROZA UJP. 2 At the time of filtration, the permeability of the monoclonal antibody in MICROZA UJP was 99.2%, and the intermembrane pressure differential was 1.3 kPa.

[0129] (Example 4) The film area of ​​the wetted part is 3 cm² 2 The same procedure as in Example 1 was followed, except that a porous hollow fiber membrane with a pore size of 0.2 μm, manufactured by Repligen, was used as a minimodule of porous hollow fiber membrane with an effective length adjusted to achieve the desired result. As a result, as shown in Figures 3, 5, and 7, the cell density, cell viability, and antibody concentration in the culture medium were approximately constant. Using hollow fiber from Repligen, the culture medium was 300 L / m³. 2 At the time of filtration, the permeability of the monoclonal antibody through hollow fibers manufactured by Repligen was 86.4%, and the intermembrane pressure differential was 1.0 kPa.

[0130] (Example 5) The film area of ​​the wetted part is 3 cm² 2 The same procedure as in Example 1 was followed, except that a porous hollow fiber membrane with a pore size of 0.45 μm, manufactured by Cytiva, was used as a minimodule of porous hollow fiber membrane with an effective length adjusted to achieve the desired result. As a result, as shown in Figures 3, 5, and 7, the cell density, cell viability, and antibody concentration in the culture medium were approximately constant. Using hollow fiber from Cytiva, the culture medium was 300 L / m³. 2 At the time of filtration, the permeability of the monoclonal antibody through the hollow fiber manufactured by Cytiva was 80.6%, and the intermembrane pressure differential was 2.0 kPa.

[0131] (Example 6) After thawing the monoclonal antibody-producing Chinese hamster ovary (CHO) cell line (ATCC CRL-12445), which had been acclimatized to serum-free medium and suspended in suspension, the cells were selected and frozen. Then, 10 mL of serum-free medium with the composition shown in Table 3 was pre-dispensed into a 125 mL Erlenmeyer flask, and the cells and medium were mixed in the flask. Cell count was confirmed using a viable and dead cell autoanalyzer (Vi-CELL XR, Beckman), and the cell density was 3.5 × 10⁶. 5 The cells were diluted with culture medium to a concentration of cells / mL. Then, the cells were cultured in an incubator at 37°C under a 5% CO2 atmosphere with shaking for 4 days. [Table 3]

[0132] Four days after thawing the cells, 3.5 × 10 5 50 mL of culture medium containing cells at a density of cells / mL was placed in two new 125 mL Erlenmeyer flasks, and the cells were cultured with shaking in an incubator at 37°C under a 5% CO2 atmosphere for 3 days. After 3 days, 3.5 × 10⁶ cells were observed. 5 120 to 130 mL of culture medium containing cells at a density of cells / mL was placed into two new 250 mL Erlenmeyer flasks, and the cells were cultured with shaking in an incubator at 37°C under a 5% CO2 atmosphere for 3 days. After 3 days, 3.5 × 10⁶ cells were observed. 5 500 mL of culture medium containing cells at a density of cells / mL was placed into three new 1 L Erlenmeyer flasks, and the cells were cultured in an incubator at 37°C under a 5% CO2 atmosphere with shaking for 3 days.

[0133] In a 12L capacity culture tank that has been pre-sterilized by autoclaving, place 5.5 x 10 5 5 L of culture medium containing cells at a density of cells / mL was aseptically added. The cells were cultured for 3 days at 37°C in a 5% CO2 atmosphere, while oxygen was blown in to ensure that the DO did not fall below 70%. After 3 days, continuous culture was started with filtration using a porous hollow fiber membrane (Asahi Kasei Medical, BioOptimal MF-SL0190, pore size 0.4 μm) and addition of culture medium to the culture vessel.

[0134] The same amount of fresh culture medium as the amount of culture medium removed from the culture tank by filtration was aseptically transferred from a 50L bag (Thermo Fisher, Productainer BioProcess Container (BPC), 50L) to the culture tank, controlling the amount of culture medium in the tank to remain constant. The culture medium exchange rate was 1 vvd -1 Set the vessel volumes per day to 0.5 vvd when the glucose concentration falls below 1 g / L or the glutamine concentration falls below 1 mmol / L. -1 They began to separate.

[0135] Seven days after starting continuous culture, 1 × 10 8 The culture medium containing cells at a density of cells / mL was aseptically collected, and 600 mL of the culture medium was transferred to a spinner flask, which had been pre-sterilized by autoclaving, to serve as a culture vessel. The membrane area of ​​the wetted part was 3 cm². 2 A mini-module of a porous hollow fiber membrane (Asahi Kasei Medical, BioOptimal MF-SL, pore size 0.4 μm) was prepared with an adjusted effective length to achieve the desired result. The spinner flask and the first opening of the hollow section on the primary side of the porous hollow fiber membrane were connected by a first channel, the second opening of the hollow section of the porous hollow fiber membrane and the spinner flask were connected by a second channel, and the secondary side of the porous hollow fiber membrane and the spinner flask were connected by a third channel.

[0136] The ambient temperature of the spinner flask and porous hollow fiber membrane was set to 4°C, and the culture medium was stirred in the spinner flask. The culture medium was delivered from the spinner flask to the porous hollow fiber membrane via a first channel using a magnetic levitation centrifugal pump (Levitronix PuraLev i30SU) and tangential flow filtration was performed. The culture medium that passed through the hollow portion of the porous hollow fiber membrane without being filtered was returned to the spinner flask via a second channel. The culture medium filtered through the porous hollow fiber membrane was returned to the spinner flask via a third channel. The first and third channels were designed to allow sampling of the culture medium inside.

[0137] Single-use pressure gauges (PendoTECH, PREPS-N-000 or PREPS-N-012) were installed on the primary and secondary sides of the porous hollow fiber membrane, and the transmembrane pressure difference (TMP) was measured over time. The viscosity of the culture medium was measured with an EMS viscometer (EMS-1000S), and the amount of culture medium delivered from the spinner flask to the porous hollow fiber membrane was determined when the shear stress on the inner surface of the porous hollow fiber membrane was 1.69 N / m 2 The settings were configured as follows: The filtration flow rate in the porous hollow fiber membrane was set to 5 μL / min using a pump to maintain a constant flow rate (1 LMH).

[0138] The culture medium circulating between the spinner flask and the porous hollow fiber membrane was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. As shown in Figures 4, 6, and 8, the cell density, cell viability, and antibody concentration in the culture medium remained approximately constant.

[0139] (Example 7) The film area of ​​the wetted part is 3 cm² 2 The same procedure as in Example 6 was followed, except that a porous hollow fiber membrane with a pore size of 0.2 μm, manufactured by Asahi Kasei and using Microza UMP, was used as a minimodule of porous hollow fiber membrane with an effective length adjusted to achieve the desired result. As a result, as shown in Figures 4, 6, and 8, the cell density, cell viability, and antibody concentration in the culture medium remained approximately constant.

[0140] (Example 8) The film area of ​​the wetted part is 3 cm² 2 The same procedure as in Example 6 was followed, except that a porous hollow fiber membrane with a pore size of 0.65 μm, manufactured by Asahi Kasei, was used as a minimodule of porous hollow fiber membrane with an effective length adjusted to achieve the desired result. As a result, as shown in Figures 4, 6, and 8, the cell density, cell viability, and antibody concentration in the culture medium remained approximately constant.

[0141] (Example 9) The film area of ​​the wetted part is 3 cm² 2The same procedure as in Example 6 was followed, except that a porous hollow fiber membrane with a pore size of 0.2 μm, manufactured by Repligen, was used as a minimodule of porous hollow fiber membrane with an effective length adjusted to achieve the desired result. As a result, as shown in Figures 4, 6, and 8, the cell density, cell viability, and antibody concentration in the culture medium remained approximately constant.

[0142] (Example 10) The film area of ​​the wetted part is 3 cm² 2 The same procedure as in Example 6 was followed, except that a porous hollow fiber membrane with a pore size of 0.45 μm, manufactured by Cytiva, was used as a minimodule of porous hollow fiber membrane with an effective length adjusted to achieve the desired result. As a result, as shown in Figures 4, 6, and 8, the cell density, cell viability, and antibody concentration in the culture medium remained approximately constant.

[0143] (Comparative Example 1) Similar to the example, 3.5 × 10 5 Place 120-130 mL of culture medium containing cells at a density of cells / mL into two new 250 mL Erlenmeyer flasks. Incubate the cells in an incubator at 37°C under a 5% CO2 atmosphere with shaking for 3 days. Then transfer the cells to a pre-autoclaved 3 L culture vessel, accumulating 5.5 × 10⁶ cells. 5 A 1.28 L culture medium containing cells at a density of cells / mL was aseptically added. The cells were cultured for 3 days at 37°C in a 5% CO2 atmosphere, while blowing in oxygen to ensure that the DO did not fall below 70%. After 3 days, the membrane surface area of ​​the wetted portion was 200 cm². 2 Continuous culture was initiated using a porous hollow fiber membrane module (Asahi Kasei Medical, BioOptimal MF-SL, pore size 0.4 μm) with adjusted number and effective length, followed by filtration and addition of culture medium to the culture tank.

[0144] The culture tank and the first opening of the hollow section on the primary side of the porous hollow fiber membrane were connected by a first flow path, and the second opening of the hollow section of the porous hollow fiber membrane was connected to the culture tank by a second flow path. The culture medium filtered by the porous hollow fiber membrane was not returned to the culture tank. The culture tank was also provided with an outlet for sampling the culture medium inside and a supply port for supplying fresh culture medium.

[0145] The same amount of fresh culture medium as the amount of culture medium removed from the culture tank by filtration was aseptically transferred from a 20L bag (Thermo Fisher, Productainer BioProcess Container (BPC), 20L) to the culture tank, controlling the amount of culture medium in the tank to remain constant. The culture medium exchange rate was 1 vvd -1 Set the vessel volumes per day to 0.375vvd when the glucose concentration falls below 1g / L or the glutamine concentration falls below 1 mmol / L. -1 They began to separate.

[0146] The temperature of the culture medium in the culture tank was controlled to 37°C, and the culture medium was agitated within the tank. A magnetic levitation centrifugal pump (Levitronix PuraLev 200MU) was used to transfer the culture medium from the culture tank to a porous hollow fiber membrane via a first channel, and tangential flow filtration was performed. The culture medium that passed through the hollow portion of the porous hollow fiber membrane without being filtered was returned to the culture tank via a second channel. When the DO of the culture medium fell below 70%, oxygen was introduced into the culture medium in the culture tank using a sparger. In addition, air containing 5% carbon dioxide was continuously introduced into the culture tank.

[0147] Single-use pressure gauges (PendoTECH, PREPS-N-038) were installed on the primary and secondary sides of the porous hollow fiber membrane, and the transmembrane pressure difference (TMP) was measured over time. The amount of culture medium delivered from the culture tank to the porous hollow fiber membrane was determined when the shear stress on the inner surface of the porous hollow fiber membrane was 2.10 N / m 2The settings were configured as follows: The filtration flow rate in the porous hollow fiber membrane was gradually increased by a pump to 1.0 LMH, 1.5 LMH, 2.0 LMH, 2.5 LMH, and 3.0 LMH, and the amount of fresh medium introduced was increased to adjust the glucose and glutamine concentrations in accordance with the increase in cells. In addition, the cell density in the culture medium in the culture vessel was 7.5 x 10⁶. 7 The culture was bled to a cell / mL level. Furthermore, a liquid level sensor was placed in the culture tank, and fresh culture medium was introduced into the tank to maintain a constant liquid level.

[0148] The culture medium in the culture vessel was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. As shown in Figure 9, the cell density in the culture medium continued to increase until bleeding began. As shown in Figure 10, the cell viability remained approximately constant. As shown in Figure 11, the antibody concentration tended to increase.

[0149] (Comparative Example 2) The film surface area of ​​the wetted part is 200 cm². 2 The same procedure as in Comparative Example 1 was followed, except that a porous hollow fiber membrane with a pore size of 0.2 μm, manufactured by Asahi Kasei, was used as the module for the porous hollow fiber membrane, with the number and effective length adjusted to achieve the desired result.

[0150] The culture medium in the culture vessel was sampled once or twice a day, and the cell density, cell viability, and antibody concentration in the culture medium were measured. As shown in Figure 9, the cell density in the culture medium continued to increase until bleeding began. As shown in Figure 10, the cell viability remained approximately constant. As shown in Figure 11, the antibody concentration tended to increase.

[0151] (Examples 1 to 3) 1.5 × 10⁻⁶ prepared in the same manner as in Example 1 7 The culture medium containing cells at a density of cells / mL was aseptically collected, and 600 mL of the culture medium was transferred to a spinner flask, which had been pre-sterilized by autoclaving, to serve as a culture vessel.

[0152] The ambient temperature of the spinner flask was set to 4°C, and the culture medium was agitated within the spinner flask. The spinner flask was not connected to a hollow fiber membrane; cells were cultured only within the spinner flask. The culture medium in the spinner flask was sampled once or twice daily, and the cell density, cell viability, and antibody concentration were measured. Experiments were also conducted under the same conditions except that the ambient temperature was set to room temperature (RT), and under the same conditions except that the ambient temperature was set to 37°C. As shown in Figure 12, cell density tended to increase at room temperature. As shown in Figure 13, cell viability tended to decrease at both room temperature and 37°C. As shown in Figure 14, antibody concentration tended to increase at both room temperature and 37°C.

[0153] (Reference examples 4 to 6) 1.5 × 10⁻⁶ prepared in the same manner as in Example 1 7 The culture medium containing cells at a density of cells / mL was aseptically collected, and 600 mL of the culture medium was transferred to a spinner flask, which had been pre-sterilized by autoclaving, to serve as a culture vessel.

[0154] The ambient temperature of the spinner flask was set to 8°C, and the culture medium was agitated within the spinner flask. No hollow fiber membrane was connected to the spinner flask; cells were cultured only within the spinner flask. The culture medium in the spinner flask was sampled once or twice daily, and the cell density, cell viability, and antibody concentration were measured. Experiments were also conducted under the same conditions except for an ambient temperature of 12°C, and under the same conditions except for an ambient temperature of 37°C. As shown in Figure 15, the cell density tended to decrease at 37°C. As shown in Figure 16, the cell viability tended to decrease at 37°C.

[0155] (Reference examples 7 and 8) 1.5 × 10⁻⁶ prepared in the same manner as in Example 1 7 The culture medium containing cells at a density of cells / mL was aseptically collected, and 600 mL of the culture medium was transferred to a spinner flask, which had been pre-sterilized by autoclaving, to serve as a culture vessel.

[0156] The ambient temperature of the spinner flask was set to 4°C, and the culture medium was agitated within the spinner flask. No hollow fiber membrane was connected to the spinner flask; cells were cultured only within the spinner flask. The culture medium in the spinner flask was sampled once or twice daily, and the cell density, cell viability, and antibody concentration were measured. The experiment was also conducted under the same conditions except that the culture medium was circulated using a pump. A magnetic levitation centrifugal pump (Levitronix PuraLev i30SU) was used to deliver the culture medium from the spinner flask to the pump via a first channel. The culture medium that passed through the pump was returned to the spinner flask via a second channel. The pumping speed of the culture medium was 50 mL / min. The first channel had a structure that allowed for sampling of the culture medium inside. The culture medium in the spinner flask was sampled from the first channel once or twice daily, and the cell density, cell viability, and antibody concentration were measured. As a result, as shown in Figures 17 to 19, the temporal changes in the culture medium were suppressed both with and without circulation of the culture medium by a pump.

[0157] (Analysis method) The analytical methods used in the above-mentioned examples, comparative examples, and reference examples are described below. The cell density and viability in the culture medium were measured using a live-dead cell autoanalyzer (Vi-CELL XR, Beckman Coulter). Samples were diluted with PBS(-) (Fujifilm Wako) and 600 μL were used for analysis. The image analysis method used was "CHO" in Vi-CELL XR, and the settings for Minimum diameter (μm), Cell brightness (%), and Viable cell spot brightness (%) were changed as appropriate to match the actual conditions.

[0158] Antibody concentration was measured by HPLC using the following method. (1) Detector: UV absorbance spectrophotometer (measurement wavelength: 280 nm) (2) Column: POROS G 20μm Column, 4.6×50mm, 0.8mL (ThermoFisher) (3) Column temperature: Room temperature (4) Mobile phase Mobile phase A: 7.098 g of disodium hydrogen phosphate (anhydrous) and 8.766 g of sodium chloride were dissolved in 800 mL of water, 1 mol / L hydrochloric acid was added to adjust the pH to 7.0, and then water was added to make a total volume of 1000 mL. Mobile phase B: 12 ​​mL of 1 mol / L hydrochloric acid and 8.766 g of sodium chloride were dissolved in water to make a total volume of 1000 mL. (5) Delivery of the mobile phase The fluid was delivered at a flow rate of 2 mL / min, with the ratio of mobile phase A to mobile phase B varied as shown in Table 4 below. [Table 4]

[0159] The cell culture medium was centrifuged at 300 × g for 2 minutes, and the supernatant was sampled. Using the method described above, nine serial dilutions of commercially available human immunoglobulin G (Venoglobulin IH 5% intravenous injection 2.5 g / 50 mL, manufactured by the Japan Blood Products Organization) and the cell culture medium supernatant were delivered using the procedure described above. A standard curve was created using nine peak areas of human immunoglobulin G, and the antibody concentration in each solution was calculated from the standard curve and the peak areas of the samples.

[0160] The shear stress SS at the wetted surface of the hollow fiber membrane due to the culture medium is given by the product of the viscosity VC (Pa·s) of the culture medium flowing through the hollow portion of the hollow fiber membrane and the shear rate SV ( / s), as shown in the following formula. SS = VC × SV

[0161] The shear rate can be calculated from the linear velocity and the channel diameter, and the linear velocity can be obtained from the pump output. Therefore, the shear stress was calculated based on the viscosity of the culture medium and the shear rate calculated from the linear velocity. [Explanation of Symbols]

[0162] 11, 111... Culture tanks, 12, 112... Hollow fiber membranes, 13, 14, 15, 113, 114, 115, 116, 117, 118, 119... Flow channels, 16... Agitators, 23, 25, 123, 125, 126... Pumps, 33, 34, 35, 133, 134, 135... Pressure gauges, 50... Temperature control tanks, 201... Containers, 216... Culture medium tanks, 217... Containers, 218... Containers

Claims

1. Under conditions of an ambient temperature of 15°C or lower and a culture medium temperature of 15°C or lower, the culture medium containing cells is sent from a culture tank to a porous membrane, the culture medium that has passed through the porous membrane without being filtered is returned to the culture tank, and the culture medium is circulated between the culture tank and the porous membrane. To evaluate one or more filtration conditions in the porous membrane, including, Method for evaluating filtration conditions.

2. A method for evaluating filtration conditions according to claim 1, wherein the culture medium that has passed through the porous membrane without being filtered is returned to the culture tank, and the culture medium that has passed through the porous membrane without being filtered and the culture medium that has been filtered by the porous membrane are returned to the culture tank.

3. The method further includes evaluating the filtration performance of the porous membrane under one or more of the aforementioned filtration conditions, A method for evaluating filtration conditions according to claim 1, comprising evaluating one or more filtration conditions in the porous membrane based on the filtration performance of the porous membrane.

4. The method for evaluating filtration conditions according to claim 1, wherein the progression of the cell cycle is suppressed under at least one of the conditions of an ambient temperature of 15°C or lower and a culture medium temperature.

5. The method for evaluating filtration conditions according to claim 1, wherein the proliferation of the cells is suppressed under at least one of the conditions of an ambient temperature of 15°C or lower and a culture medium temperature.

6. The method for evaluating filtration conditions according to claim 5, wherein at least one of the ambient temperature of 15°C or less and the culture medium temperature is at least one of the ambient temperature of 10°C or less and the culture medium temperature of 5°C or less.

7. The method for evaluating filtration conditions according to claim 1, wherein the enzyme activity of the cells is suppressed under at least one of the conditions of an ambient temperature of 15°C or lower and a culture medium temperature.

8. The method for evaluating filtration conditions according to claim 7, wherein the enzyme is a cyclin-dependent kinase.

9. The method for evaluating filtration conditions according to claim 1, wherein a cell cycle progression inhibitor is present in the culture medium.

10. A method for evaluating filtration conditions according to claim 1, comprising evaluating one or more filtration conditions in the porous membrane based on the permeability of the cell products in the porous membrane.

11. A method for evaluating filtration conditions according to claim 1, comprising evaluating one or more filtration conditions in the porous membrane based on the permeation flux in the porous membrane.

12. A method for evaluating filtration conditions according to claim 1, comprising evaluating one or more filtration conditions in the porous membrane based on the intermembrane pressure difference in the porous membrane.

13. A method for evaluating filtration conditions according to claim 1, comprising evaluating one or more filtration conditions in the porous membrane based on the turbidity of the filtrate.

14. The method for evaluating filtration conditions according to claim 1, wherein the one or more filtration conditions are one or more conditions for the structure, material, or physical properties of the porous membrane.

15. The method for evaluating filtration conditions according to claim 1, wherein the one or more filtration conditions are one or more conditions of the culture medium.

16. The method for evaluating filtration conditions according to claim 1, wherein the one or more filtration conditions are one or more conditions for the density of the cells in the culture medium.

17. The method for evaluating filtration conditions according to claim 1, wherein the one or more filtration conditions are one or more conditions for the flow rate of the culture medium sent to the porous membrane.

18. The method for evaluating filtration conditions according to claim 1, wherein the one or more filtration conditions are one or more conditions for the flow rate of the culture medium filtered by the porous membrane.

19. The method for evaluating filtration conditions according to claim 1, wherein the one or more filtration conditions are one or more conditions for shear stress on the liquid-contacting surface of the porous membrane due to the flow of culture medium supplied to the porous membrane.

20. The method for evaluating filtration conditions according to claim 1, wherein the culture medium, including the culture tank and the porous membrane, is circulated within a temperature-controlled tank.

21. The method for evaluating filtration conditions according to claim 1, wherein no active operation is performed to maintain the composition of the culture medium when circulating the culture medium between the culture tank and the porous membrane.

22. A method for evaluating filtration conditions according to claim 1, wherein the culture solution is circulated between the culture tank and the porous membrane, and no culture medium is added from the outside to the path through which the culture solution circulates, including the culture tank and the porous membrane.

23. A method for evaluating filtration conditions according to claim 1, wherein the culture medium is circulated between the culture tank and the porous membrane, and at least one of the dissolved oxygen and pH of the culture medium is not controlled.

24. A method for evaluating filtration conditions according to claim 1, wherein, in circulating the culture medium between the culture tank and the porous membrane, the cells are not bred from the pathway through which the culture medium circulates, including the culture tank and the porous membrane.

25. The method for evaluating filtration conditions according to claim 1, further comprising circulating the culture solution between the culture tank and the porous membrane, and taking a sample of the culture solution from the path through which the culture solution circulates, including the culture tank and the porous membrane.

26. The method for evaluating filtration conditions according to claim 25, further comprising measuring the density of the cells in the sampled culture medium.

27. The method for evaluating filtration conditions according to claim 25, further comprising measuring the viability of the cells in the sampled culture medium.

28. The method for evaluating filtration conditions according to claim 25, further comprising measuring the concentration of the cell products in the sampled culture medium.

29. The method for evaluating filtration conditions according to claim 25, further comprising measuring the turbidity of the culture medium in the sampled culture medium.

30. The method for evaluating filtration conditions according to claim 1, wherein the porous membrane is a hollow fiber membrane.

31. The method for evaluating filtration conditions according to claim 1, wherein the porous membrane is a microfiltration membrane.

32. Under conditions of an ambient temperature of 15°C or lower and a culture medium temperature, the culture medium containing cells is sent from a culture tank to a porous membrane, the culture medium that has passed through the porous membrane without being filtered is returned to the culture tank, the culture medium is circulated between the culture tank and the porous membrane, and one or more filtration conditions in the porous membrane are evaluated to obtain filtration conditions. Under conditions in which cells proliferate, the filtration conditions are used to send the culture medium containing the cells from the culture tank to the porous membrane, return the culture medium that has passed through the porous membrane without being filtered to the culture tank, recover the culture medium containing the cell products filtered by the porous membrane, and circulate at least a portion of the culture medium between the culture tank and the porous membrane. including, Method for producing cell products.

33. The method for producing cell products according to claim 32, wherein, when evaluating one or more filtration conditions in the porous membrane, the culture medium that has passed through the porous membrane without being filtered is returned to the culture tank, and both the culture medium that has passed through the porous membrane without being filtered and the culture medium that has been filtered by the porous membrane are returned to the culture tank.

34. The method for producing a cell product according to claim 32, wherein the conditions for the proliferation of the cells are the conditions for the progression of the cell cycle.

35. The method for producing cell products according to claim 32, wherein the conditions for the proliferation of the cells are at least one of an ambient temperature of 15°C or higher and a culture medium temperature.

36. The method for producing cell products according to claim 35, wherein at least one of the ambient temperature of 15°C or higher and the culture medium temperature is at least one of the ambient temperature of 20°C, 25°C, 30°C, or 35°C.

37. The method for producing a cell product according to claim 32, wherein the conditions for the proliferation of the cells are conditions in which the enzymes of the cells are activated.

38. The method for producing a cell product according to claim 37, wherein the enzyme is a cyclin-dependent kinase.

39. The method for producing cell products according to claim 32, wherein the condition for the proliferation of the cells is the absence of a cell cycle inhibitor in the culture medium.

40. A method for producing cell products according to claim 32, wherein the progression of the cell cycle is suppressed under at least one of the conditions of an ambient temperature of 15°C or lower and a culture medium temperature.

41. The method for producing cell products according to claim 32, wherein the proliferation of the cells is suppressed under at least one of the conditions of an ambient temperature of 15°C or lower and a culture medium temperature.

42. The method for producing cell products according to claim 41, wherein at least one of the ambient temperature of 15°C or less and the culture medium temperature is at least one of 10°C or less and 5°C or less.

43. The method for producing cell products according to claim 32, wherein the enzyme activity of the cells is suppressed under at least one of the conditions of an ambient temperature of 15°C or lower and a culture medium temperature.

44. The method for producing a cell product according to claim 43, wherein the enzyme is a cyclin-dependent kinase.

45. The method for producing cell products according to claim 32, wherein a cell cycle progression inhibitor is present in the culture medium when one or more filtration conditions in the porous membrane are evaluated.

46. A method for producing cell products according to claim 32, wherein one or more filtration conditions in the porous membrane are evaluated based on the permeability of the cell products in the porous membrane.

47. A method for producing cell products according to claim 32, wherein one or more filtration conditions in the porous membrane are evaluated based on the permeation flux in the porous membrane.

48. A method for producing cell products according to claim 32, wherein one or more filtration conditions in the porous membrane are evaluated based on the intermembrane pressure difference in the porous membrane.

49. A method for producing cell products according to claim 32, wherein one or more filtration conditions in the porous membrane are evaluated based on the turbidity of the filtrate.

50. The method for producing cell products according to claim 32, wherein the one or more filtration conditions are one or more conditions for the structure, material, or physical properties of the porous membrane.

51. The method for producing cell products according to claim 32, wherein the one or more filtration conditions are one or more conditions of the culture medium.

52. The method for producing cell products according to claim 32, wherein the one or more filtration conditions are one or more conditions for the density of the cells in the culture medium.

53. The method for producing cell products according to claim 32, wherein the one or more filtration conditions are one or more conditions for the flow rate of the culture medium sent to the porous membrane.

54. The method for producing cell products according to claim 32, wherein the one or more filtration conditions are one or more conditions for the flow rate of the culture medium filtered through the porous membrane.

55. The method for producing cell products according to claim 32, wherein the one or more filtration conditions are one or more conditions for shear stress on the liquid-contacting surface of the porous membrane due to the flow of culture medium supplied to the porous membrane.

56. The method for producing cell products according to claim 32, wherein, when evaluating one or more filtration conditions in the porous membrane, the culture vessel and the culture medium including the porous membrane are arranged in a temperature control tank.

57. A method for producing cell products according to claim 32, wherein, when evaluating one or more filtration conditions in the porous membrane, no active operation is performed to maintain the composition of the culture medium when circulating the culture medium between the culture tank and the porous membrane.

58. The method for producing cell products according to claim 32, wherein, when evaluating one or more filtration conditions in the porous membrane, the culture medium is circulated between the culture tank and the porous membrane, and no culture medium is added from the outside to the path through which the culture medium circulates.

59. The method for producing cell products according to claim 32, wherein, when evaluating one or more filtration conditions in the porous membrane, the culture medium is circulated between the culture tank and the porous membrane, and at least one of the dissolved oxygen and pH of the culture medium is not controlled.

60. A method for producing cell products according to claim 32, wherein, when evaluating one or more filtration conditions in the porous membrane, the culture medium is circulated between the culture tank and the porous membrane, and the cells are not bred from the pathway through which the culture medium circulates, including the culture tank and the porous membrane.

61. A method for producing cell products according to claim 32, wherein, when evaluating one or more filtration conditions in the porous membrane, the culture medium is circulated between the culture tank and the porous membrane, and the method for producing cell products according to claim 32 includes taking a sample of the culture medium from a path through which the culture medium circulates, including the culture tank and the porous membrane.

62. The method for producing a cell product according to claim 61, further comprising measuring the density of the cells in the sampled culture medium.

63. The method for producing cell products according to claim 61, further comprising measuring the viability of the cells in the sampled culture medium.

64. The method for producing cell products according to claim 61, further comprising measuring the concentration of the cell products in the sampled culture medium.

65. The method for producing cell products according to claim 61, further comprising measuring the turbidity of the culture medium in the sampled culture medium.

66. The method for producing cell-derived organisms according to claim 32, wherein the porous membrane is a hollow fiber membrane.

67. The method for producing cell-derived organisms according to claim 32, wherein the porous membrane is a microfiltration membrane.

68. The method for producing a cell product according to claim 32, wherein the cells are perfused and cultured.

69. Under conditions where a cell cycle progression inhibitor is present in the culture medium, the culture medium containing the cells is sent from a culture tank to a porous membrane, the culture medium that has passed through the porous membrane without being filtered is returned to the culture tank, and the culture medium is circulated between the culture tank and the porous membrane. To evaluate one or more filtration conditions in the porous membrane, including, Method for evaluating filtration conditions.

70. Under conditions in which a cell cycle progression inhibitor is present in the culture medium, the culture medium containing cells is sent from a culture tank to a porous membrane, the culture medium that has passed through the porous membrane without being filtered is returned to the culture tank, the culture medium is circulated between the culture tank and the porous membrane, and one or more filtration conditions in the porous membrane are evaluated to obtain the filtration conditions. Under conditions in which cells proliferate, the filtration conditions are used to send the culture medium containing the cells from the culture tank to the porous membrane, return the culture medium that has passed through the porous membrane without being filtered to the culture tank, recover the culture medium containing the cell products filtered by the porous membrane, and circulate at least a portion of the culture medium between the culture tank and the porous membrane. including, Method for producing cell products.

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